Investigation of the effects of deracoxib and piroxicam on the in vitro viability of osteosarcoma cells from dogs

OBJECTIVE: To determine whether exposure of canine osteosarcoma cells to deracoxib or piroxicam results in decreased viability, whether the cytotoxic effects of deracoxib and piroxicam involve induction of apoptosis, and whether deracoxib is a more potent inhibitor of osteosarcoma cell growth than piroxicam. SAMPLE POPULATION: 1 fibroblast and 3 osteosarcoma cell lines. PROCEDURE: Cell counts and viability assays were performed using osteosarcoma cells (POS, highly metastatic POS, and canine osteosarcoma cell 31) and fibroblasts after 72 hours of incubation with deracoxib at concentrations of 0.5 microM to 500 microM or piroxicam at concentrations of 1 microM to 1,000 microM. Percentage viability was determined for each concentration. A DNA fragmentation analysis was performed to assess drug-induced apoptosis. RESULTS: Concentration of deracoxib required for 50% inhibition of cell viability (IC50) was reached in all 3 osteosarcoma cell lines and ranged from 70 to 150 microM, whereas the IC50 for piroxicam was only reached in the POS cell line at 500 microM. Neither deracoxib nor piroxicam induced sufficient toxicity in fibroblasts to reach an IC50. Exposure of osteosarcoma cells to cytotoxic concentrations of deracoxib and piroxicam did not result in DNA fragmentation. CONCLUSIONS AND CLINICAL RELEVANCE: Intermediate and high concentrations of deracoxib and high concentrations of piroxicam were cytotoxic to osteosarcoma cells; neither drug inhibited cell viability at typical plasma concentrations in dogs. Deracoxib inhibited viability of cells at concentrations that did not affect fibroblast viability. There was no evidence of apoptosis induction for either drug; however, only 1 cell line was evaluated for apoptosis induction and only for a limited selection of drug concentrations.     

Results of the Combined Treatment with Piroxicam and Carboplatin in Canine Oral Non‐Tonsillar Squamous Cell Carcinoma

Introduction:  Over‐expression of COX‐2 has been observed in several human and animal malignancies and is implicated in carcinogenesis through the conversion of arachidonic acid to PGE‐2. Use of platinum‐containing cytostatic agents and/or (non‐)specific COX‐2 inhibitors, has been reported as a treatment option for canine oral non‐tonsillar squamous cell carcinomas (ONT‐SCC). However, no study describes the effect of a combination of carboplatin and piroxicam on this tumor type.
Methods:  7 dogs with a T3 (WHO‐TNM) ONT‐SCC were treated with piroxicam and carboplatin. Five had bone involvement and no detectable metastasis. Two dogs without bone involvement had metastasis in the regional lymph nodes. Piroxicam was given orally 0.3 mg/kg s.i.d. Each dog was scheduled to receive between 6 and 12 carboplatin infusions (300 mg/m² i.v.) at 3 week intervals. Ondansetron and metoclopramide were used as anti‐emetic agents. The dogs are planned to receive piroxicam on a lifelong basis.
Results:  Complete response (CR) without adjuvant surgery was achieved in 4 of the 7 dogs. Two dogs needed adjuvant surgery to achieve CR. One dog had progressive disease and was euthanised 231 days after start of therapy. All the others were still alive and in CR at date of analysis. Median follow‐up was 335 days (107–689 days).
Conclusions:  Our study suggests that a combination of piroxicam and carboplatin is a useful treatment option for canine ONT‐SCC. All dogs tolerated therapy well and the 57% response rate for reaching a complete and durable remission without adjuvant surgery is promising.     

Antitumor Activity of Mycobacterial Cell Wall‐DNA Complex (MCC) Against Canine Urinary Bladder Transitional Cell Carcinoma Cells

Introduction:  Mycobacterial cell wall‐DNA complex (MCC) is a bifunctional anticancer agent that induces cancer cell apoptosis and stimulates cytokine synthesis by immune cells. Intravesical MCC is currently being evaluated in humans with high‐grade urinary bladder cancer. Evaluation of MCC in dogs with transitional cell carcinoma (TCC) will allow mechanistic studies in a natural animal model of TCC, and a potentially beneficial therapy for dogs with this cancer. In this study, we have determined the anticancer activity of MCC against canine TCC cells in vitro .
Methods:  Canine TCC cells (K9TCC cell line) were incubated with MCC (0.05–100 μg/ml, 0.5–72 hours). Cellular proliferation was measured by MTT reduction. Cell cycle was analyzed by flow cytometry with propidium iodide. Apoptosis was identified by flow cytometry using anti‐active‐caspase‐3/PE and anti‐cleaved‐PARP/FITC antibodies. Apoptosis‐inducing activity of 100 μg/ml MCC in combination with piroxicam (0.1–1.0 uM) was evaluated.
Results:  MCC inhibited K9TCC cell proliferation in a concentration‐dependent manner (maximal activity – 45% at 100 μg/ml MCC) in association with the presence of activated caspase‐3 and cleaved PARP. Inhibition of proliferation and apoptosis‐inducing activities of MCC were independent of cell cycle phase. A thirty‐minute exposure of MCC was sufficient for optimal activity. Piroxicam (0.5 uM) enhanced apoptosis‐inducing activity of MCC.
Conclusions:  MCC induces apoptosis in K9TCC cells. This activity is potentiated by piroxicam. Following positive results in vitro , in vivo studies have been initiated. One dog, treated to date, has had a minor reduction in tumor volume following the first course of treatment with no treatment‐related toxicity.     

Effects of Mycobacterial Cell Wall‐DNA Complex (MCC), Alendronate and Pamidronate on Canine Osteosarcoma Cell Lines

Introduction:  MCC, a cell wall composition prepared from Mycobacterium phlei ., inhibits the proliferation and induces apoptosis in a wide range of tumor cells. Bisphosphonates have been reported to inhibit the proliferation of canine osteosarcoma cell lines. In this study, we have determined the activity of MCC alone and in combination with the bisphosphonates alendronate and pamidronate on canine osteosarcoma cell lines.
Methods:  Canine osteosarcoma cell lines D17 and D22 were incubated with different concentrations of MCC (0.01–100 μg/ml) and suboptimal concentrations of alendronate and pamidronate for 72 hours. Cellular proliferation was measured by MTT reduction. Nuclear DNA condensation was determined using with Hoescht 33258 staining, and apoptosis by flow cytometry using active‐caspase‐3/PE and anti‐cleaved‐PARP/FITC antibodies.
Results:  MCC inhibited the proliferation of both canine osteosarcoma D17 and D22 cell lines in a concentration‐dependent manner. The IC₅₀ for D17 cells was 3.9 μg/ml and for D22 cells 44.4 μg/ml. Cells incubated with 100 μg/ml MCC were positive for Hoescht staining, active caspase‐3 and cleaved PARP, indicative of cell death by apoptosis. The addition of alendronate or pamidronate was found to potentiate the apoptosis‐inducing activity of MCC. Maximal activity was observed when 5 μM alendronante or 10 μM pamidronate were used in combination with 100 μg/ml MCC.
Conclusion:  MCC inhibits the proliferation and induces apoptosis in canine osteosarcoma cell lines in vitro . This anticancer activity can be potentiated by the use of alendronate and pamidronate. These data support the development of MCC as a therapeutic agent for the treatment of canine osteosarcoma.     

Anti‐Tumor Effect of Adenovirus‐Mediated P53 Gene Therapy on the Growth of Canine Osteosarcoma Transplanted into Nude Mice

Introduction:  It has been reported that 40–50% of canine osteosarcoma cases have p53 mutations. The p53 tumor supressor gene plays a central role in cell cycle regulation and induction of apoptosis. We previously showed that adenoviral vector expressing canine P53 (AxCA‐cp53) inhibited growth of cultured canine osteosarcoma cell lines. Here, we evaluated anti‐tumor effect of adenovirus‐mediated p53 gene therapy on the growth of canine osteosarcomas transplanted into nude mice.
Methods:  Nine nude mice were subcutaneously injected with cells of a canine osteosarcoma cell line (POS) having p53 gene mutation. The transplanted tumors formed into nude mice were injected with AxCA‐cp53, AxCA‐LacZ (adenovirus vector expressing LacZ) or PBS (3 mice each) 7 times during 15 days. Tumor sizes were measured every 3 days for 27 days after injection with the adenovirus vectors. Expression efficiency of the adenovirus‐mediated gene transfer was examined by X‐gal staining and P53 immunostaining. Effects of the P53 expression on cell cycle control were examined by RT‐PCR for expression of p21 gene downstream of P53.
Results:  Significant differences in the tumor size was observed between the transplanted osteosarcoma tissues injected with AxCA‐cp53 and those injected with AxCA‐LacZ or PBS. Expressions of LacZ and P53 were confirmed at the injection sites of the tumors. Moreover, p21 mRNA expression was shown to be induced in the AxCA‐cp53‐injected tumors, indicating the funciton of P53 to induce cell cycle arrest.
Conclusions:  Adenoviral vector expressing canine P53 inhibited the growth of canine ostersarcoma transplanted into nude mice.     

Characterization, Expression and Function of c‐MET in Canine Spontaneous Cancers

Introduction:  Aberrant expression of the proto‐oncogene c‐Met has been noted in a variety of human cancers. In dogs, inappropriate Met expression has been identified in canine osteosarcoma (OSA) tumor samples. To better define the potential role of Met dysregulation in canine cancer, we cloned canine Met, HGF, and HGF activator and evaluated their expression patterns in a variety of canine tumor cell lines.
Methods:  Canine Met, HGF, and HGF activator were cloned from normal canine liver and canine OSA cell lines using primers based on regions of homology between mouse and human sequences as well as 5' and 3' RACE.
Results:  Inappropriate expression of Met was found in canine cell lines derived from OSAs, mast cell tumors, histiocytic sarcomas, hemangiosarcoma, and melanomas. Both HGF and HGF activator were found to be expressed in several of these tumor cell lines, providing evidence of a possible autocrine loop of Met stimulation. Incubation of canine tumor cell lines with rhHGF resulted in Met autophosphorylation and activation of the downstream signaling elements Gab1, Akt and Erk1/2. Scattering of tumor cells in response to HGF occurred under conditions of cell stress, such as serum starvation. Lastly, the Met inhibitor PHA‐665752 blocked HGF induced phosphorylation of canine Met and Gab1.
Conclusions:  These studies provide evidence that similar to the case in human tumors, aberrant Met expression may play an important role in the biology of canine cancer. As such, inhibition of Met function may represent a potentially useful novel therapeutic approach.     

COX‐2 Expression in Feline Oral Squamous Cell Carcinoma (FOSCC)– An Immunohistochemical Study and Analysis of Survival

Introduction:  Cyclooxygenase‐2 (COX‐2) catalyses the rate‐limiting step in the conversion of arachidonic acid to eicosanoids and has been found to be overexpressed in many human and some animal cancers. Overexpression of COX‐2 in head and neck SCC in humans is associated with shorter survival times. Non‐resectable, FOSCC is a devastating disease with no effective therapy. Overexpression of COX‐2 in FOSCC may support the anecdotal use of NSAID therapy. Identification of a prognostic marker in cats may permit more effective, targeted therapy.
Methods:  Immunohistochemistry was performed on formalin‐fixed, paraffin‐embedded tissue from 59 FOSCC cases using an indirect staining technique and feline foetal kidney as positive control. Polyclonal antiserum specific to COX‐1 and COX‐2 were used after antigen retrieval with pH6 citrate buffer. Retrospective, observational data were collected by practitioner questionnaires. A Kaplan‐Meier survival plot was derived.
Results:  55/59 questionnaires were returned (93% response rate). Median age at presentation was 10.9 years (range 7–15.5). Median survival time was 44 days (95% CL: 31, 79). Preliminary results show that COX‐1 staining was positive in all tumour tissues and in 86.7%(13/15 cases) of normal tissues. COX‐2 staining was positive in 67.3%(37/55) of tumour tissues and absent in normal tissue. Results of proportional hazards regression for survival and multiple logistic regression for COX expression including age, sex, breed, prior NSAID administration, other medication and COX expression as potential explanatory variables will be presented.
Conclusions:  These results indicated that COX‐1 and COX‐2 expression is seen in FOSCC but may not be predictive for survival.     

Induction of Chemoresistance by Transfection of the Full‐Length Canine mdr1 Gene in Canine Cell Lines

Introduction:  In the chemotherapy for treatment of lymphoid tumors in dogs, myelosuppression is a frequently encountered dose‐limiting factor. One possible approach to overcome myelosuppression is induction of chemoresistance in hematopoietic stem cells through expression of the mdr1 gene. A full‐length canine mdr1 cDNA clone was isolated in our laboratory. The present study was carried out to assess whether it confers multidrug resistance in canine cell lines.
Materials and methods:  The full‐length canine mdr1 cDNA was inserted into an expression plasmid vector. A canine mammary tumor cell line (CIPP) and osteosarcoma cell line (OOS) were transfected with the canine mdr1 expression vector. Expression of P‐gp was examined by immunoblotting. Function of ATP‐dependent drug efflux was measured by flow cytometric analysis using Rhodamine 123. Sensitivity to chemotherapeutic drugs was shown by estimation of 50% inhibitory concentrations (IC₅₀) of vincristine or doxorubicin.
Results:  Immunoblotting of the transfected cells revealed a strong band of P‐gp detected by a monoclonal antibody directed to P‐gp. Rhodamine 123 efflux test showed an apparent drug efflux activity in the transfected cells. From the IC₅₀ of the chemotherapeutic agents, the transfected cells showed a remarkable increase (20 to 60‐fold) in the resistance to vincristine or doxorubicin.
Conclusion:  Transfection of canine mdr1 gene induced P‐gp expression and strong drug resistance in canine cell lines.     

Xenogeneic Human Tyrosinase DNA Vaccination Induces Specific Immune Response in Dogs with Advanced Malignant Melanoma

Introduction:  Xenogeneic melanosomal differentiation antigens, delivered in the form of a plasmid DNA vaccine, can overcome host immune ignorance/tolerance in preclinical animals to melanoma by: 1) generating humoral and cytotoxic T cell responses and 2) inducing protection from tumor challenge. Initial trials of human tyrosinase (huTyr) DNA vaccination of dogs with advanced malignant melanoma (Bergman et al , 2003) demonstrated safety and prolongation of survival with this therapeutic modality. We investigated antigen‐specific immunity in dogs receiving huTyr DNA vaccination.
Methods:  Three cohorts of three dogs each with advanced (WHO stage II‐IV) canine malignant melanoma (CMM) received four biweekly IM injections (dose levels 100, 500, or 1,500 ug, respectively/vaccination) of huTyr plasmid DNA via the Biojector2000 jet delivery device. Sera samples were taken before and after vaccination to detect specific antibody formation to huTyr by Enzyme‐Linked ImmunoSorbent Assays (ELISAs) and flow‐cytometry.
Results:  Three dogs have measurable, 2 to 4 fold huTyr‐specific antibody titers. Preliminary studies by flow‐cytometry have confirmed antibody response to huTyr by positive binding to endogenous human tyrosinase in SK‐Mel188 cells using post‐vaccinate serum.
Conclusions:  Xenogeneic (huTyr) DNA vaccination generates antigen‐specific humoral responses, which may partially explain the previously reported clinical efficacy and anti‐tumor responses. Ongoing studies include: 1) a comprehensive analysis by flow‐cytometry to detect huTyr‐specific antibodies and 2) a quantitative measure of potential cytotoxic T‐cell responses in these dogs by the DNA vaccination.     

Identification of a Splice Variant of gp100 Expressed in Canine Melanoma Tumours

Introduction:  The melanoma‐associated antigen (MAA) gp100 is a glycoprotein expressed by melanocytes and is considered to be an important target for anti‐tumour immunity in human melanoma. The aim of the current study was to characterise the canine gp100 gene with a view to its inclusion in a DNA vaccine for treatment of canine oral malignant melanoma.
Methods:  RNA was extracted from eight canine oral melanomas and seven control samples (pigmented oral mucocutaneous tissue) and cDNA synthesised. PCR was performed using human gp100 primers designed to generate a 421base pair (bp) fragment. In addition to the expected pcr product, a smaller amplicon (∼260 bp) was present in all tumours and control tissues. Both PCR amplicons were cloned and sequenced. The gp100 genomic DNA sequence was identified from the dog genome resource (NCBI).
Results:  The 421 bp fragment of canine gp100 shared 90.8% identity with human gp100. This partial sequence was located on dog chromosome 10 (CFA10_contig 2982). Further analysis, using the human gp100 gene as a reference, allowed the complete canine gp100 coding sequence to be identified from the dog genome. Sequence analysis of the small PCR product showed it to be a splice variant of gp100 with exon 5 deleted.
Conclusion:  The canine gp100 sequence has been identified and this will allow DNA vaccine constructs containing this MAA to be developed. The splice variant of gp100 with exon 5 deleted was not tumour‐specific and was expressed in both melanomas and normal pigmented tissue.     

Cyclooxygenase ‐2 Expression in Mammary Tumors in Dogs and its Correlation to Histologic and Biologic Behavior

Introduction:  Cyclooxygenase‐2 (Cox‐2) is the inducible form and the rate‐limiting enzyme, for conversion of arachidonic acid to prostaglandins. Cox‐2 overexpression, common in carcinomas, is associated with increased growth rate, resistance to apoptosis, angiogenesis, and overall, both local and distant aggressive behavior. Cox‐2 overexpression has been detected in human and canine mammary tumors (MTs). Histopathology of canine MT is not always predictive of biologic behavior, and anecdotally, only 50% of the malignant MTs are expected to metastasize. We hypothesize that Cox‐2 expression correlates with aggressive behavior.
Methods:  This retrospective study evaluated 48 bitches, presented for excision of MT between 2000 and 2003 at FMVZ de Botucatu‐UNESP, Brasil. Follow‐up varied from 18 months to 24 months and included physical examination and thoracic radiographs. Histopathologic examination was performed in all tumors, as well as in metastatic lesions when detected in the follow‐up period. Immunohistochemistry was used to detect expression of Cox‐2 in paraffin blocks (Rabbit polyclonal anti‐PGHS‐2. Oxford Biomedical). 10 adenomas, 10 carcinomas, 10 benign mixed tumors, 10 malignant mixed tumors and 8 cases of primary carcinomas and their metastatic lesions.
Results:  Expression of Cox‐2 varied among groups. Adenomas (32.1%), mixed benign tumors (38%), carcinomas (60.3%), malignant mixed tumors (65.8%), and metastatic carcinomas (81.25%) and their metastatic lesions (84.35%). Statistically significant differences (p < 0.05) were observed between the benign and malignant counterparts and between carcinomas and metastatic carcinomas.
Conclusions:  Cox‐2 expression correlates with both histologic and biologic behavior in mammary carcinomas, and may serve as a predictor of metastatic potential.     

Characterization of the Biological Behavior of Appendicular Osteosarcoma in Rottweilers with a Comparison to Other Breeds

Introduction:  The clinical impression of many oncologists is that Rottweilers with appendicular osteosarcoma (OSA) have an unfavorable prognosis despite standard therapy (amputation and chemotherapy). The purpose of this study was to compare clinical characteristics, biological behavior and outcome of OSA in Rottweilers to that of other breeds.
Methods:  107 OSA cases were studied: 56 clinical cases (17 Rottweilers) and 54 necropsy submissions (13 Rottweilers) of OSA (3 cases overlapping). Information regarding signalment, prognostic factors, treatment and outcome was obtained from medical records and follow‐up with owners. Data were analyzed by chi‐square test, t‐test, and log rank test.
Preliminary Results:  Compared to other breeds Rottweilers were more likely to have femur (23% vs. 16%) and ulna (15% vs. 3%) and less likely to have tibia (8% vs. 27%) as their primary tumor location (p = 0.04). In the necropsy group, Rottweilers had a younger age at death (6.4 vs. 10 years, p = 0.0002) and a greater body weight at diagnosis (49 vs. 37 kg, p = 0.002). No statistical differences in the age at diagnosis or survival time were noted in Rottweilers vs. other breeds. Metastasis to lymph nodes (31% vs. 10%, p = 0.08) and lungs (64% vs. 49%, p = 0.24) was more frequent in Rottweilers. There was no difference in chemotherapy drugs given; however, Rottweilers were 20 times more likely to receive NSAIDs (p = 0.0004).
Conclusion:  Greater body weight with a predilection for certain tumor locations may contribute to OSA development in Rottweilers. A younger age at death may suggest a more aggressive phenotype.     

Investigating the in vitro interactive effects of ionizing radiation and pamidronate in a canine osteosarcoma cell line

Introduction: Dogs with osteosarcoma suffer intense bone pain. Conventional palliative treatment options include ionizing radiation with or without systemic chemotherapy. Intravenous pamidronate is a first‐line therapeutic agent in people with painful skeletal metastases and is currently being evaluated for the management of osteolytic pain associated with canine osteosarcoma. The theoretical combination of radiation and pamidronate for managing bone pain appears to be a rational treatment option, however, in vitro experiments demonstrating additive or supraadditve cytotoxic effects of these two modalities would further support the clinical institution of this novel combination therapy. The purpose of this study was to evaluate the in vitro cytotoxicity of ionizing radiation and pamidronate in a canine OSA cell line and to characterize the interaction between these two therapeutic modalities when used in combination. Methods: The canine osteosarcoma cell line, HMPOS, was subjected independently to varying doses of ionizing radiation (100 cGy, 300 cGy, 400 cGy, 600 cGy, 800 cGy, or 1000 cGy) or different concentrations of pamidronate (10, 25, 50, 66, 75, 100, 150, 200, and 250 μM). Cells were harvested following 48 hours of incubation and cell viability was assessed by flow cytometric analysis. Dose‐response curves were generated, and a theoretical ED₅₀ for each treatment modality calculated. An ED₅₀ isobologram was created to determine if different combinations of ionizing radiation and pamidronate (isoboles) would demonstrate subadditive, additive, or supraadditve cytotoxic effects. Results: The ED₅₀ of ionizing radiation and pamidronate were 614 cGy and 65.3 μM, respectively. Of the six different isobole combinations evaluated, three produced supraadditve effects, one produced a subadditive effect, and two produced additive effects. Conclusions: Both ionizing radiation and pamidronate independently exert in vitro cytotoxic effects. Positive interactive effects can be generated, but only with specific isobole combinations. The results from this in vitro study support the use of specific combinations of ionizing radiation and pamidronate in order to maximize the cytotoxic properties of both treatment modalities.     

RT‐PCR based Tyrosine Kinase Display Profiling of Canine Melanoma: IGF‐1 Receptor as a Potential Therapeutic Target

Introduction:  There is a wealth of information available regarding tyrosine kinase (TK) expression in human cancer, however there is minimal information regarding the expression and function of TKs in canine melanoma, and no attempt has been made to systematically define the repertoire of TKs expressed. This study employed a molecular technique called RT‐PCR display to simultaneously evaluate the expression of up to 30 different TKs in a canine melanoma cell line.
Materials and Methods:  mRNA was extracted and reverse‐transcribed from the 17CM98 canine melanoma line, then subjected to PCR using degenerate primers coding for highly conserved regions which flank the kinase domains of many receptor and nonreceptor TKs. The resulting product was ligated into a plasmid vector and used to transform E. coli . Multiple colonies were isolated, and the cDNA inserts sequenced.
Results and Conclusions:  Sequencing 46 clones yielded canine homologs of IGF‐1R (50%), JAK1 (17%), PDGFR‐α(11%), FGFR1 (9%), Axl (7%), c‐Abl (4%), and PTK2 (2%). Interestingly, IGF‐1R, JAK1 and Axl were detected using a similar technique in human melanoma, supporting the cross‐species validity of this assay. Given the abundance of IGF‐1R clones, we sought to determine the biologic effect of rhIGF‐1 in 17CM98 cells. IGF‐1 stimulated IGF‐1R phosphorylation, cell proliferation and VEGF production in 17CM98, and protected the cells from serum starvation‐induced apoptosis. Expression of IGF‐1R mRNA was detected in 5 of 5 additional canine melanoma cell lines evaluated, suggesting that IGF‐1R expression may be common in canine melanoma cells and providing a novel target for future therapy.     

Biological activity of dihydroartemisinin in canine osteosarcoma cell lines

OBJECTIVE: To evaluate the biological activity of dihydroartemisinin on canine osteosarcoma cell lines in vitro. SAMPLE POPULATION: 4 canine osteosarcoma cell lines. PROCEDURES: Cell viability assays were performed on canine osteosarcoma cell lines OSCA2, OSCA16, OSCA50, and D17 after 24, 48, and 72 hours of treatment with dihydroartemisinin at concentrations of 0.1 to 100 microM. Apoptosis was assessed by use of an ELISA for free nuclosomal DNA fragmentation and by western blot analysis for cleavage of caspase 3. Cell cycle analysis was performed by use of staining with propidium iodide and flow cytometry. Detection of reactive oxygen species (ROS) was conducted in the D17 cell line by use of 6-carboxy-2',7'-dihydrofluorescein diacetate and flow cytometry. RESULTS: The concentration of dihydroartemisinin required for 50% inhibition of cell viability (IC50) was achieved in all 4 canine osteosarcoma cell lines and ranged from 8.7 to 43.6 microM. Induction of apoptosis was evident as an increase in nucleosomal DNA fragmentation, cleavage of caspase 3, and an increase in the population in the sub G0/G1 phase of the cell cycle detected by flow cytometry. Exposure to dihydroartemisinin also resulted in a decrease in the G0/G1 population. Iron-dependent generation of ROS was detected in dihydroartemisinin-treated D17 cells; ROS generation increased in a dose-dependent manner. CONCLUSIONS AND CLINICAL RELEVANCE: Incubation with dihydroartemisinin resulted in biological activity against canine osteosarcoma cell lines, which included induction of apoptosis and arrest of the cell cycle. Clinical trials of dihydroartemisinin in dogs with osteosarcoma should be conducted.     

The Utility of Flow Cytometry for the Diagnosis of Cranial Mediastinal Malignancy in Canine Patients

Introduction:  The most common neoplasms located in the anterior mediastinum in the canine are thymoma and lymphoma. Distinguishing between the two is a diagnostic challenge. Treatment and prognosis for these diseases differs significantly. Thymomas contain a population of normally developing T cells. The majority of these T cells exhibit an immature phenotype, characterized by co‐expression of CD4 and CD8. This phenotype is rarely seen on neoplastic lymphocytes. The purpose of this study was to determine if analysis by flow cytometry could discriminate thymoma from lymphoma based on these cell surface markers.
Methods:  Fine needle aspirates were obtained from ten canine patients with mediastinal masses. Cells were analyzed by flow cytometry using a panel of T and B cell markers.
Results:  Six cases with 10% or greater CD4 + CD8+ cells were diagnosed with thymoma and were confirmed by histopathology. Four cases had fewer than 5% CD4 + CD8+ cells, having lymphocytes expressing CD4 only (3 cases) or CD21, a B cell marker(1 case). These were confirmed as lymphoma by cytology and/or a clonality assay. The sensitivity and specificity of this assay when used in the diagnostic work‐up for suspected thymoma was 100%.
Conclusion:  Flow cytometry may provide important, complementary information in the diagnostic work‐up of the canine patient with a mediastinal mass.     

The in vitro effects of piroxicam and meloxicam on canine cell lines

OBJECTIVES: To analyse the direct antiproliferative effects of both piroxicam and meloxicam at a variety of concentrations on a series of canine cancer cell lines and the mechanism of cell death. METHODS: The in vitro effects of piroxicam and meloxicam at various concentrations on canine cell cultures (Madin-Darby canine kidney cells, osteosarcoma, mammary carcinoma, and lymphoma) were assessed with respect to proliferation inhibition and apoptosis induction. Western blot analysis of cyclooxygenase-1 and cyclooxygenase-2 expression was performed on all cell lines. RESULTS: All cell lines used in this study were cyclooxygenase-1 and cyclooxygenase-2 positive apart from Madin-Darby canine kidney cells which were negative for both cyclooxygenase-1 and cyclooxygenase-2. Both meloxicam and piroxicam were able to inhibit proliferation in cell lines in a dose-dependent manner. However, the drug concentration required for a given effect was cell line dependent. CLINICAL SIGNIFICANCE: The results suggest that significant inhibition of proliferation and induction of apoptosis would only occur when drug concentrations were in excess of those that can be achieved in vivo following maximum recommended dose rates. It is possible, however, that local or topical treatment or altered dosing regimens may offer alternative approaches to the use of these drugs as antineoplastic agents.     

Retropective Evaluation of Survival Time of Dogs with Stage III Osteosarcoma that Undergo Treatment

Introduction:  Stage III osteosarcoma generally carries a grave prognosis. Despite this, some owners elect to treat using palliative or curative intent protocols. The purpose of this study was to determine the survival times for dogs with stage III osteosarcoma that undergo treatment and to evaluate the variables that affect survival time.
Methods:  Retrospective study using the CSU Animal Cancer Center osteosarcoma data base. Search criteria included dogs diagnosed with osteosarcoma 1985–2004 with metastasis at the time of presentation. Dogs were excluded if they were euthanized at the time of diagnosis (13 dogs) or lost to follow‐up (10 dogs). There were 90 cases for analysis. The survival times were compared based on the primary tumor site, site of metastasis, treatment given, age, sex and breed.
Results:  Survival times in days ranged from 0 to 1583 days. The overall median survival time was 76 days. The one‐year, two‐year and three‐year percent survival were 7%, 4.7% and 3.5%, respectively. Treatment included various combinations of chemotherapy, surgery, radiation therapy, bisphosphonates and NSAIDs. Findings included an increased survival time with surgery and adjuvant therapy compared with surgery alone and a decreased survival time with metastasis to the lung or lymph node.
Conclusions:  Treatment of dogs with stage III osteosarcoma can result in variable survival times. Multimodality therapy appears to result in longer survival times. Metastasis to the lung or lymph node correlated with a decreased survival time.